Mitochondria, the site of a variety of important metabolic processes, are essential organelles of eukaryotic organisms. Pathological effects of reduced bioenergetic capacity and altered iron metabolism caused by mitochondrial dysfunction are common in human populations. Molecular chaperones play a vital role in the biogenesis of mitochondria. The goal of this proposal is to understand the mechanism of action of Hsp70/J-protein molecular chaperones in two critical mitochondrial processes - translocation of proteins from the cytosol into the mitochondrial matrix and the generation of Fe/S clusters, critical co- factors for numerous enzymes. The vast majority of the hundreds of proteins of the mitochondrial matrix are synthesized on cytosolic ribosomes. Thus, efficient import of proteins is critical for mitochondrial function. The import motor required for driving proteins across the inner membrane into the matrix is composed of 5 essential components, with the matrix Hsp70, Ssc1, at its core. We will concentrate on regulatory mechanisms that increase the efficiency of the import motor. We will use genetic, biochemical and structural approaches, with a goal of understanding the specialized regulated protein:protein interactions that have evolved to drive efficient translocation of proteins across the membrane. The mitochondrial matrix contains a set of essential proteins devoted to the biogenesis of Fe/S clusters. The clusters are assembled on the scaffold protein, Isu, prior to transfer to recipient apo- proteins. The J-protein:Hsp70 chaperone pair, Jac1:Ssq1, binds to Isu and facilitates transfer of the cluster. To understand the mechanism of this chaperone function in Fe/S cluster biogenesis the temporal set of interactions between Isu and other proteins required for Fe/S cluster formation and transfer will be determined, using biochemical interaction assays and exploiting mutant proteins having defects in their interactions with partner proteins. The yeast mitochondrial system will also be used as a model to understand the molecular basis of the specialization of Hsp70s. Knowledge gained as to the basis of the specialization of multiple Hsp70s of the mitochondrial matrix will serve as a paradigm for understanding how Hsp70s have evolved to function in an array of physiological processes in other cellular compartments particularly in the case of the less well-defined human Hsp70 family. PUBLIC HEALTH RELEVANCE: The research described in this proposal focuses on understanding fundamental aspects of mitochondrial function and biogenesis. Mitochondria are essential organelles that are vital for energy production. Reduced mitochondrial function has been linked to a wide array of health issues from age-related neurological and cardiovascular disease to early onset neuromuscular disorders.